A comparison between biochemical tests and the 'CAMP' reaction for identification of Aeromonas spp.

Aeromonas bacteria (110 strains) from a variety of clinical, food and environmental sources, were identified using routine biochemical tests. Concurrently they were tested aerobically and anaerobically for their ability to perform synergistic haemolysis with Staphylococcus aureus (the 'CAMP' reaction). Results did not support a reported observation that the 'CAMP' reaction can he used to facilitate speciation of Aeromonas bacteria.     

Incidence and identification of mesophilic Aeromonas spp. from retail foods

Sixty-eight food samples were examined for the presence of mesophilic Aeromonas species both qualitatively and quantitatively. Aeromonads were isolated from 26% of the vegetable samples, 70% of the meat and poultry samples and 72% of the fish and shrimps. Numbers of motile aeromonads present in the food samples varied from <10(2) cfu g(-1) to >10(5) cfu g(-1). GLC analysis of FAMEs was used to identify a selection of presumptive Aeromonas colonies to fenospecies or genomic species level. Aeromonas strains belonging to the Aer. caviae complex, which also includes the potentially pathogenic genospecies HG4, were mostly isolated from vegetables but were also found in meat, poultry and fish. In addition, three strains of the virulent taxon Aer. veronii biovar sobria HG8 were isolated from poultry and minced meat. All members of the Aer. hydrophila complex, predominant in the fish, meat and poultry samples, were classified in the non-virulent taxon HG3. Although the significance of Aeromonas in foods remains undefined, the isolation of Aeromonas HG4 and HG8 strains from a variety of retail foods may indicate that these products can act as possible vehicles for the dissemination of food-borne Aeromonas gastroenteritis.     

Comparison of three rapid methods for identification of Salmonella spp

A study was carried out to compare three rapid methods for detection of Salmonella spp. The fluorogenic MUCAP test (Biolife, Italy), the SM-ID agar test (bioMérieux, France) and the Rambach agar test (Merck, Germany) were used in this study to examine 103 strains (69 Salmonella strains and 34 non- Salmonella strains). Two conventional culture media, Hektoen and Leifson agars, were also included. The sensitivities of the MUCAP, SM-ID, Rambach and Hektoen agar tests for pure strains were 100, 93, 88 and 99%, respectively, and their specificities were 74, 97, 76 and 59%, respectively. A total of 100 stool samples from patients with acute diarrhoea was also tested and showed great discrepancy between the different methods. In agreement with other investigators, it was found that the discriminating capacity of Rambach and SM-ID as primary plating media was very restricted. The MUCAP test was very sensitive, rapid and easy to perform but not very specific. In view of these results, it is essential to combine different methods for the accurate and reliable detection of Salmonella strains.     

Polymorphism of Aeromonas spp. tRNA intergenic spacers

We investigated the length polymorphism of the intergenic spacers lying between tRNA genes of Aeromonas spp. A total of 69 strains representing all known genomic species of Aeromonas were used in the study. tDNA-PCR patterns were examined by Dice coefficient (S(D)) and unweighted pair group method of clustering (UPGMA). The strains were allocated into 15 groups at a similarity level of 70%. The strains belonging to seven genomic species: A. hydrophila (HG 1), A. caviae (HG 4), A. sobria (HG 7), A. veronii (HG 8/10), A. encheleia (HG 16), A. popoffii (HG 17), and A. culicicola (HG 18) formed distinct clusters. Our study revealed a genetic heterogeneity of the following species: A. bestiarum, A. salmonicida, A. media, A. eucrenophila, A. jandaei, A. schubertii, and A. allosaccharophila.     

PCR-based assays for the fish pathogen Aeromonas salmonicida. I. Evaluation of three PCR primer sets for detection and identification

In an effort to develop a rapid diagnostic test for the fish pathogen Aeromonas salmonicida, the performance of 2 polymerase chain reaction (PCR) primer sets (AP and PAAS) targeting the fish pathogen A. salmonicida and 1 PCR primer set (MIY) targeting A. salmonicida subsp. salmonicida were evaluated. Initially, the PCR assays were used to screen purified DNA extracted from 308 A. salmonicida isolates. The AP and PAAS PCR tests were demonstrated to be 100% specific for the species A. salmonicida and did not cross-react with any of the non-target organisms (bacterial species other than A. salmonicida) used in this study. The combined sensitivity of the AP and PAAS tests was 99.4% and offered the best coverage in terms of identifying the target organism. The MIY PCR appeared to be 100% sensitive and specific for A. salmonicida subsp. salmonicida. Studies with tissues, spiked with known quantities of bacteria, were conducted to determine the lower detection limit of the PCR tests, and then the ability of these PCR tests to detect A. salmonicida in experimentally infected salmonids was assessed.     

Evaluation of a digoxigenin-labelled probe for detection of Aeromonas spp.

An outer membrane protein-based Digoxigenin (DIG)-labelled DNA probe was developed for the specific detection of Aeromonas sp. from food/environmental/clinical samples. Dot blot reaction answered for all the Aeromonas isolates and was negative for Escherichia coli , Pseudomonas sp., Klebsiella sp., Vibrio parahaemolyticus , V. harveyi , V. alginolyticus , V. vulnificus . Edwardsiella tarda and Staphylococcus sp. As this protein is highly conserved in various Aeromonas species, the probe has the potential for use as a rapid and reliable diagnostic tool.     

Assessment of media used for selective isolation of Aeromonas spp.

A. GAVRIEL AND A.J. LAMB. 1995. A group of selective media were evaluated for their ability to support growth and recovery of type species of the mesophilic, motile group from the genus Aeromonas . With both low and high inoculum densities Aer. hydrophila and Aer. caviae grew well on ampicillin dextrin agar, glutamate starch penicillin agar and starch glutamate ampicillin penicillin agar whereas Aer. veronii only grew with a high level of inoculum. Aeromonas sobria and Aer. schubertii displayed little growth on any of these media regardless of the inoculum size. These results indicate that environmental studies attempting to evaluate the relative distribution and abundance of the members of the motile Aeromonas group would not recover all the strains with equal effectiveness using these particular media.     

Experimental Pathogenicity of Aeromonas spp. for the Zebra Mussel, Dreissena polymorpha

Experiments were conducted to determine whether species of Aeromonas were pathogenic to the zebra mussel Dreissena polymorpha. A. jandaei, A. veronii, and A. media, identified with Biolog, were originally isolated from dead zebra mussels. When inoculated into living mussels, these bacteria resulted in the mortality of the bivalves. Two additional species, A. salmonicida salmonicida (ATCC 33678) and A. hydrophila (ATCC 7966), were also demonstrated to be pathogenic to the mussels. In addition to the pathogenicity, the data also suggest that the zebra mussels may be an important reservoir for these bacteria in freshwater environments. Received: 26 March 1997 / Accepted: 7 July 1997     

Medium for the presumptive identification of Aeromonas hydrophila and Enterobacteriaceae.

A medium was devised for the rapid presumptive identification of Aeromonas hydrophila. It also offered good differentiation of Klebsiella, Proteus, and other enteric species. Mannitol fermentation, inositol fermentation, ornithine decarboxylation and deamination, indole production, motility, and H2S production from sodium thiosulfate and cysteine could be recorded in a single tube of the medium.     

Exotoxin production by Aeromonas spp. in foods

A psychrotrophic toxin-producing strain of Aeromonas hydrophila grew well in a range of food slurries (scallop, prawn, fish, chicken liver paté, liverwurst, chicken luncheon slice and commercial baby food preparations) held at refrigeration temperatures. In most foods, excluding the baby food preparations, exotoxins were produced at levels comparable with production in bacteriological broth without apparent food spoilage (all but prawn and fish). Addition of ultra-heat treated (UHT) milk to toxin-containing broth culture supernatants markedly decreased or removed haemolytic and cytotoxic activities, explaining low levels of toxins found in milk in a previous study. Baby food preparations did not inactivate exotoxins under similar conditions suggesting production of toxins rather than their inactivation was inhibited in these foods.     

Species identification of clinically important Aeromonas spp. by restriction fragment length polymorphism of 16S rDNA

AIMS: The aim of the study was to characterize 16S rDNA of Aeromonas spp. to rapidly identify clinically important species of these bacteria. METHODS AND RESULTS: Sequence analysis of published 16S rDNA for unique restriction sites revealed prospect of species identification. Extraction of genomic DNA followed by amplification and step-by-step restriction endonuclease digestion of 16S rDNA was able to identify Aeromonas spp. of medical significance. Validation of the method was performed by subjecting 53 Aeromonas strains of multiple origin to similar treatment. Results of the study were in agreement with corresponding species of the isolates. CONCLUSIONS: The method developed offers an easily interpretable tool for the identification of Aeromonas spp. of clinical relevance. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed methodology should facilitate routine laboratory diagnosis of Aeromonas spp. from clinical cases to species level.     

Biochemical identification and numerical taxonomy of Aeromonas spp. isolated from environmental and clinical samples in Spain

AIMS: To study the phenotypic characteristics of Aeromonas spp. from environmental and clinical samples in Spain and to cluster these strains by numerical taxonomy. METHODS AND RESULTS: A collection of 202 Aeromonas strains isolated from bivalve molluscs, water and clinical samples was tested for 64 phenotypic properties; 91% of these isolates were identified at species level. Aeromonas caviae was predominant in bivalve molluscs and Aerom. bestiarum in freshwater samples. Cluster analyses revealed eight different phena: three containing more than one DNA-DNA hybridization group but including strains that belong to the same phenospecies complex (Aerom. hydrophila, Aerom. sobria and Aerom. caviae), Aerom. encheleia, Aerom. trota and three containing unidentified Aeromonas strains isolated from bivalve molluscs. CONCLUSIONS:Aeromonas spp. are widely distributed in environmental and clinical sources. A selection of 16 of the phenotypical tests chosen allowed the identification of most isolates (91%), although some strains remain unidentified, mainly isolates from bivalve molluscs, suggesting the presence of new Aeromonas species. Numerical taxonomy was not in total concordance with the identification of the studied strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Numerical taxonomy of Aeromonas strains isolated from different sources revealed the presence of potentially pathogenic Aeromonas spp., especially in bivalve molluscs, and phena with unidentified strains that suggest new Aeromonas species.     

A note on Aeromonas spp. from chickens as possible food-borne pathogens

The possible role of Aeromonas spp. as potential food-borne psychrotrophic pathogens was investigated by examining organisms isolated from processed raw chicken for their biochemical characteristics, ability to produce exotoxins and to grow at chill temperatures. These strains, in particular A. sobria, with identical characteristics to human diarrhoea-associated aeromonads were readily found. Chicken, and human and environmental (water) strains characterized in a previous study, were investigated for their ability to grow at refrigeration temperatures (5 +/- 2 degrees C) and, for selected strains, the theoretical minimum temperature for growth (Tmin) was determined from the growth pattern in a temperature gradient incubator. All enterotoxigenic chicken strains tested were typical mesophiles, with an optimal growth temperature of approximately 37 degrees C and Tmin values approximately 4.5 degrees C. They were rapidly outgrown by a psychrotrophic Pseudomonas sp. typical of spoilage biota found on food. Enterotoxin was not produced below 15 degrees C by any of the toxigenic food strains tested. The Aeromonas strains isolated from chickens in this study seem unlikely therefore to be a significant health risk, provided the chickens are properly stored and cooked. This would appear to be substantiated by the lack of reports of food-associated outbreaks of illness from these sources.     

A note on Aeromonas spp. from chickens as possible food-borne pathogens

The possible role of Aeromonas spp. as potential food-borne psychrotrophic pathogens was investigated by examining organisms isolated from processed raw chicken for their biochemical characteristics, ability to produce exotoxins and to grow at chill temperatures. These strains, in particular A. sobria , with identical characteristics to human diarrhoea-associated aeromonads were readily found. Chicken, and human and environmental (water) strains characterized in a previous study, were investigated for their ability to grow at refrigeration temperatures (5 ± 2°C) and, for selected strains, the theoretical minimum temperature for growth ( T _min) was determined from the growth pattern in a temperature gradient incubator. All enterotoxigenic chicken strains tested were typical mesophiles, with an optimal growth temperature of ˜37°C and T _min values ˜4.5°C. They were rapidly outgrown by a psychrotrophic Pseudomonas sp. typical of spoilage biota found on food. Enterotoxin was not produced below 15°C by any of the toxigenic food strains tested. The Aeromonas strains isolated from chickens in this study seem unlikely therefore to be a significant health risk, provided the chickens are properly stored and cooked. This would appear to be substantiated by the lack of reports of food-associated outbreaks of illness from these sources.     

The activity of ceftiofur sodium for Aeromonas spp. isolated from ornamental fish.

Our objective was to determine the activity of ceftiofur sodium against Aeromonas hydrophila and A. sobria isolated from a variety of domestic and imported tropical fish. Twelve antimicrobial drugs were tested for effectiveness against these aeromonads using the Kirby-Bauer Disk Diffusion technique and minimum inhibitory concentration determinations. Ceftiofur sodium was highly effective in vitro against aeromonads isolated from ornamental fish. Of the 42 isolates of Aeromonas spp. tested, none were resistant to ceftiofur sodium; however, all isolates were resistant to ampicillin, and 71% were resistant to tetracycline.     

Antimicrobial Susceptibilities of Aeromonas spp. Isolated from Environmental Sources

Aeromonas spp. are ubiquitous aquatic bacteria that cause serious infections in both poikilothermic and endothermic animals, including humans. Clinical isolates have shown an increasing incidence of antibiotic and antimicrobial drug resistance since the widespread use of antibiotics began. A total of 282 Aeromonas pure cultures were isolated from both urban and rural playa lakes in the vicinity of Lubbock, Texas, and several rivers in West Texas and New Mexico. Of these, at least 104 were subsequently confirmed to be independent isolates. The 104 isolates were identified by Biolog and belonged to 11 different species. The MICs of six metals, one metalloid, five antibiotics, and two antimicrobial drugs were determined. All aeromonads were sensitive to chromate, cobalt, copper, nickel, zinc, cefuroxime, kanamycin, nalidixic acid, ofloxacin, tetracycline, and sulfamethoxazole. Low incidences of trimethoprim resistance, mercury resistance, and arsenite resistance were found. Dual resistances were found in 5 of the 104 Aeromonas isolates. Greater numbers of resistant isolates were obtained from samples taken in March versus July 2002 and from sediment versus water. Plasmids were isolated from selected strains of the arsenite- and mercury-resistant organisms and were transformed into Escherichia coli XL1-Blue MRF′. Acquisition of the resistance phenotypes by the new host showed that these resistance genes were carried on the plasmids. Mercury resistance was found to be encoded on a conjugative plasmid. Despite the low incidence of resistant isolates, the six playa lakes and three rivers that were sampled in this study can be considered a reservoir for antimicrobial resistance genes.